Cordray and Richards‑Kortum Malar J (2015) 14:472

DOI 10.1186/s12936-015-0995-6 Malaria Journal

RESEARCH Open Access

A paper and plastic device for the

combined isothermal amplification and lateral
flow detection of Plasmodium DNA
Michael S. Cordray*  and Rebecca R. Richards‑Kortum

Abstract 
Background:  Isothermal amplification techniques are emerging as a promising method for malaria diagnosis since
they are capable of detecting extremely low concentrations of parasite target while mitigating the need for infrastruc‑
ture and training required by other nucleic acid based tests. Recombinase polymerase amplification (RPA) is promising
for further development since it operates in a short time frame (<30 min) and produces a product that can be visually
detected on a lateral flow dipstick. A self-sealing paper and plastic system that performs both the amplification and
detection of a malaria DNA sequence is presented.
Methods:  Primers were designed using the NCBI nBLAST tools and screened using gel electrophoresis. Paper and
plastic devices were prototyped using commercial design software and parts were cut using a laser cutter and assem‑
bled by hand. Synthetic copies of the Plasmodium 18S gene were spiked into solution and used as targets for the RPA
reaction. To test the performance of the device the same samples spiked with synthetic target were run in parallel
both in the paper and plastic devices and using conventional bench top methods.
Results:  Novel RPA primers were developed that bind to sequences present in the four species of Plasmodium which
infect humans. The paper and plastic devices were found to be capable of detecting as few as 5 copies/µL of synthetic
Plasmodium DNA (50 copies total), comparable to the same reaction run on the bench top. The devices produce
visual results in an hour, cost approximately $1, and are self-contained once the device is sealed.
Conclusions:  The device was capable of carrying out the RPA reaction and detecting meaningful amounts of syn‑
thetic Plasmodium DNA in a self-sealing and self-contained device. This device may be a step towards making nucleic
acid tests more accessible for malaria detection.
Keywords:  Paper microfluidics, Recombinase polymerase amplification, Nucleic acid test

Background complexity of NATs, since they do not require a thermo-

There is growing interest in developing nucleic acid
tests (NATs) that can be used to diagnose and monitor
infectious diseases in low-resource areas [1, 2]. NATs
are extremely sensitive and highly specific, but their
widespread use at the point-of-care is hindered by high
per-test cost, technical complexity and infrastructure
requirements [3–5]. Isothermal amplification meth-
ods offer a promising avenue to reduce the cost and
*Correspondence: mcordray@gmail.com
Rice University Department of Bioengineering, 6100 Main St., Houston,
TX 77005, USA
cycler, thereby increasing global access to high quality
diagnostics [6–9].
Recombinase polymerase amplification (RPA) is a
recently developed isothermal amplification method that
can produce detectable results in less time and at lower
temperatures than other isothermal amplification tech-
niques; these advantages make it particularly suitable for
use at the point-of-care. The RPA reactions operates in a
manner similar to conventional PCR, using a forward and
reverse primer to amplify a sequence of DNA. However
instead of using heat to denature the target strand, the
RPA reaction operates isothermally by using enzymes.

© 2015 Cordray and Richards-Kortum. This article is distributed under the terms of the Creative Commons Attribution 4.0 Inter‑
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mons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecom‑
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Cordray and Richards‑Kortum Malar J (2015) 14:472
Recombinase is added to the master mix, which forms
complexes with the primer strands. These recombinase-
primer complexes then displace the anti-sense strand of
the target, binding the primers to the target strands and
creating binding sites for polymerase to bind [10]. The
RPA reaction can be modified (RPA nfo) with an internal
probe sequence and a labelled primer to generate a prod-
uct which can be detected by a commercial lateral flow
dipstick assay, allowing for visual readout of the result
[11, 12].
Paper microfluidic devices offer a promising platform
to translate isothermal amplification tests to the point-
of-care, since they are made of inexpensive materials,
do not require pumps or other external apparatus and
can replicate many of the functions of more traditional
microfluidic devices [13, 14]. Recently, Rohrman et  al.
presented a foldable paper and plastic microfluidic device
to carry out the RPA reaction [15]. This system was used
to amplify a target sequence in the HIV gag gene, and
achieved detectable amplification of as few as 10 copies
of HIV DNA in as few as 15  min. However a key limi-
tation of this system was that it required the user to cut
the device open to extract and dilute the amplified prod-
uct for detection on lateral flow strips. Without dilution,
the crowding reagent used in the RPA nfo buffer leads to
false positive results on the lateral flow detection strips.
The need to cut open the device and dilute the reaction
products adds additional user steps and greatly increases
the likelihood of contamination of the workspace with
the amplified target, which in turn can lead to false posi-
tive results in future tests.
Here, an integrated device capable of carrying out
isothermal amplification using the RPA reaction, post-
amplification dilution, and lateral flow detection of the
resulting product is presented. This device is self-sealing
and once loaded and sealed contains all reagents and
mechanisms necessary to amplify, dilute and detect the
product without the need to open the system.
As an example of a disease which would benefit from
increased access to NAT-based diagnostics, this inte-
grated system was used to detect synthetic copies of a
gene present in Plasmodium, the parasite which causes
malaria [16]. There is a need for increased access to
point-of-care malaria diagnostics which can detect the
low-level infections which are often missed by thin and
thick smear microscopy, the current gold standard [17–
19]. Several isothermal amplification techniques have
been investigated as a method for diagnosing malaria,
but RPA nfo offers advantages due to its short run time
and easy visual detection scheme [20–27]. RPA nfo prim-
ers and a probe were developed which target a region
common to all human infectious species of Plasmodium.
The integrated paper and plastic system can be used to
Page 2 of 8
detect clinically relevant amounts of a synthetic copy of
the Plasmodium 18S gene.
Methods
Materials
RPA nfo amplification kits and lateral flow detec-
tion strips (Milenia Hybridtech 1) were obtained from
TwistDx Limited (Cambridge, UK). DNA primer, probe
and target plasmid sequences were obtained from Inte-
grated DNA Technologies (Coralville, IA, USA) and
all except the plasmid prepared by suspending them in
water to a concentration of 10  µM. Materials for plas-
mid cloning and purification were ordered from Qia-
gen Inc. (Valencia, CA, USA). Sheets of 0.003″ acetate
(KD3CL0811) and double-stick tape (KDT912-12) were
obtained from Grafix (Maple Heights, OH, USA). Blot-
ter paper (CFSP223000) and glass fiber (GFCP203000)
pads were obtained from Millipore Corp (Billerica, MA,
USA). Whatman 1 Chromatography paper was obtained
from Fisher Scientific Company LLC (Pittsburgh, PA,
USA). Agarose, TAE, ethidium bromide, TBST and water
were obtained from Sigma Aldrich (St. Louis, MO, USA).
Screw top microcentrifuge tubes (used for all bench top
reactions) were obtained from Genesee Scientific Corp
(San Diego, CA, USA). 25 g metal weights were obtained
from Amazon (Seattle, WA, USA).
Primer design and screen
Primers and a probe specific to the Plasmodium 18S gene
were designed based on guidelines provided by TwistDx,
the manufacturer of the RPA reaction, and were modi-
fied with end-tags and internal modifications to make
them compatible with lateral flow detection. The target
selected is the 18S gene because it is highly conserved,
and contains regions shared amongst all species of Plas-
modium which commonly infect humans [28, 29]. For
testing purposes, the target used was a synthetic plasmid
containing a 2090 bp copy of the malaria 18S gene (total
size of plasmid 5091 bp) (GenBank: MS19172). This plas-
mid was cloned into E. coli to increase copy number and
purified using standard protocols [30]. Successful cloning
and purification of the gene was confirmed by gel elec-
trophoresis on a 3 % agarose gel stained by ethidium bro-
mide. For all experiments, this purified target was then
diluted in water to a concentration of 200, 50 and 5 cop-
ies/µL before being input to RPA nfo reactions along with
an additional no target control of water.
The NCBI’s nucleotide BLAST tools were used to
search for primers specific to Plasmodium without sig-
nificant overlap with other genomes. A variety of forward
and reverse primer candidates were chosen and pairs
were screened by observing their performance on a 3 %
agarose gel stained with ethidium bromide and imaged
Cordray and Richards‑Kortum Malar J (2015) 14:472
on a Bio-Rad Gel Doc XR+. Once the outer primer pairs
were chosen, internal probes were screened through
a similar process. The set of primers and probe with
the brightest band on the gel were selected for further
experimentation:
Forward primer: 5′-CACGAACTAAAAACGGCCATG
CATCACCATCC-3′
Reverse primer: 5′-biotin-CCTTATGAGAAATCAAA
GTCTTTGGGTTCTGGGG-3′
Probe: 5′-FAM-ATCAAGAAAGAGCTATTAATCTG
TCAATCCTAC-THF-CTTGTCTTAAACTAGTG-
SpC3-3′
The selected primers and probe target a sequence pre-
sent in the genome of all major species of Plasmodium,
which infect humans, based on BLAST searches of the
NCBI nucleotide database. This set of primers and probe
produce a 218  bp primary product labelled only with
biotin, as well as a 181  bp secondary product which is
labelled for detection on the lateral flow strips.
To generate a product detectable on lateral flow strips,
the 5′ end of the reverse primer is labelled with a biotin.
A probe was designed that is labeled on its 5′ end with
a FAM group, a C3 spacer (SpC3) on the 3′ end and a
tetrahydrofuran residue (THF) which replaces an inter-
nal base. The RPA reaction produces a primary prod-
uct which is labelled with a biotin tag from the reverse
primer. The probe binds internally to this primary prod-
uct, and once bound, the nfo enzyme in the reaction mix
removes the THF and the bases 3′ to it, including the C3
spacer. This allows the remaining bases of the probe to
act as a new forward primer that produces a secondary
product labelled with both biotin and FAM. This second-
ary product was detected using lateral flow strips func-
tionalized with streptavidin to capture the product at
the test line and gold nanoparticles functionalized with
anti-FAM to produce a color change at the test line in the
presence of the labelled secondary product.
RPA reaction in solution
The RPA nfo reaction was carried out following the man-
ufacturer’s recommended protocols [10]. In brief, a mas-
ter mix was created containing 2.1 µL each of 10 µM of
each primer, 0.6 µL of 10 µM of the probe, 3.2 µL of water
and 29.5 µL of the reaction buffer included with the kit.
In all experiments 37.5  µL of master mix was aliquoted
into individual reaction tubes containing the freeze-
dried reaction pellet included with the kit. To each of
these tubes 10  µL of target was added, and then 2.5  µL
of 280 mM magnesium acetate (MgAc) was added to the
cap of the reaction tube. Tubes were then briefly centri-
fuged and vortexed to mix MgAc with the other reagents.
Page 3 of 8
The reaction tubes were then incubated for 30  min in a
heat block at 37 °C.
Lateral flow detection and imaging
Immediately after incubation, RPA reactions were
removed from heat and 2  µL was removed and added
to 98  µL of tris-buffered saline with 0.05  % tween-20
(TBST), which was used as both sample diluent and
lateral flow running buffer. This dilution step is neces-
sary because the dextran sulfate used as a crowding
reagent in the RPA nfo buffer causes non-specific bind-
ing of the antibody labelled gold to the test line, generat-
ing a false positive. After dilution, 10  µL of the product
was removed and spotted onto the end of a lateral flow
strip. The strip was placed in a well containing 98 µL of
TBST and allowed to run for 5 min. The strips were then
scanned using a commercial document scanner (Epson
Perfection V500 Photo) and images were recorded at
1200 dpi using the ‘reflective document’ settings.
The signal-to-background ratio (SBR) of the lateral flow
images were analysed using a custom MATLAB script.
The user manually segments the test line and the sur-
rounding background region. The SBR is calculated by
calculating the ratio of the average intensity of the signal
and background regions [31].
RPA reaction in paper and plastic device
Device description
The paper and plastic device needs to carry out three
main functions: (1) amplify the target using RPA; (2)
dilute the resulting product; and (3) detect the prod-
uct using a lateral flow sandwich assay. In addition, the
device must transfer the product between the ampli-
fication, dilution, and detection modules. A sequence
of paper pads loaded with various reagents was used to
carry out these functions. The amplification reaction is
carried out on the RPA pad (Fig. 1A): a rectangular piece
of Whatman number 1 paper which holds the RPA nfo
reagents. The dilution function is carried out by four
dilution pads (small squares of blotter paper), in two
separate stages of dilution (Fig. 1B, C). Detection is car-
ried out using a commercial lateral flow strip (Fig.  1D)
with running buffer delivered via a running buffer pad.
To move amplified product from the amplification zone
to each of the two dilution zones and then to the lateral
flow strip, the sample pad (Fig.  1E), is physically moved
across the other parts of the device. When the sample
pad is placed in contact with another pad, passive diffu-
sion results in target transport to a subsequent zone of
the device. To improve mixing during the amplification
step, an extra pad of glass fiber is placed so that it will
be in contact with the sample pad when the sample pad
is in contact with the RPA pad. This mixing pad (Fig. 1F)
Cordray and Richards‑Kortum Malar J (2015) 14:472
Fig. 1  Layout and components of the paper and plastic amplifica‑
tion and detection device. The device is shown in its configuration
just before it would be loaded with wet reagents. The sample sliders
are shown off to the side for clarity. The slots where they would be
inserted, which are cut into the hinge where the device folds shut,
are highlighted with an oval outline. The components of the device are
A the RPA pad; B the dry dilution pads; C the wet dilution pads; D the
lateral flow strip; E the sample pad; F the mixing pad; G the base layer;
H the acetate mask; I the sample slider; J the running buffer pad; and
K the running buffer slider
helps promote the diffusion of the RPA master mix onto
the sample pad.
The case of the device is made of a symmetric L-shaped
piece of acetate backed double-stick tape (Fig.  1G).
The RPA pad, dilution pads and lateral flow strip are
all attached to this base layer. The RPA pad is placed at
the top of the L, followed by pairs of dilution pads along
the vertical section of the L, and the lateral flow strip is
placed along the base of the L. An acetate mask (Fig. 1H)
is adhered to the remaining exposed adhesive of the base
layer to prevent the device from adhering to itself when
folded shut for use.
A slider is used to move the sample pad through the
system. The slider holds the sample pad between two
layers of acetate-backed tape with a hole cut through it
to expose the surface of the sample pad. The slider has a
long tail which sticks out through a slot in the base layer
and is pulled to slide the sample pad between the dif-
ferent functional parts of the device (Fig.  1I). A second
Page 4 of 8
slider holds a running buffer pad (running buffer pad
Fig.  1J, running buffer slider Fig.  1K), which is used to
deliver running buffer to the lateral flow strip.
Device assembly
All device components were cut using a 60 W CO2 laser
cutter (Universal Laser Systems, Scottsdale, AZ, USA).
One side of a double stick tape sheet was exposed and
adhered to acetate prior to cutting. Acetate backed pieces
of tape were cut with contact paper side up. The fol-
lowing settings were used for each material: acetate 3 %
power, 10  % speed; blotter paper 5  % power, 5  % speed;
Whatman 1 paper 3 % power, 5 % speed; glass fiber 3 %
power, 5  % speed; and cuts through the double-stick
tape 7.5 % power, 10 % speed. After the acetate and tape
base layer was cut, the outlines for each component were
scored using a setting that cuts only through the contact
paper (1.8 % power, 12 % speed).
To assemble the device, the contact paper was removed
from the scored areas and the four dilution pads and the
lateral flow strip were adhered to the base layer. The RPA
nfo reaction pellet was placed on the exposed tape and
the RPA pad pressed down on top to crush the pellet. The
remaining paper (except for the separate border piece)
was removed and the acetate mask was adhered to the
base. The sample and running buffer sliders were assem-
bled. The tails of the finished sliders were threaded into
the slots cut in the base layer.
Operation of device
To operate the device, the remaining border of contact
paper is removed to expose the sticky edge that will be
used to seal the device closed. The device is then loaded
with wet reagents necessary for amplification, dilution
and detection. 100  µL of TBST is added to the running
buffer pad. An additional 40 µL of TBST is added to the
two wet dilution pads. 37.5 µL of the RPA nfo master mix
is added to the pad covering the RPA nfo pellet. Finally
10 µL of the sample is added to the sample slider, along
with 2.5 µL of MgAc. The device is then folded in half and
sealed shut.
The sample slider begins centered over the RPA pad,
position 1, so that when the device is sealed shut, the
sample and RPA reagents mix together. To begin ampli-
fication, the entire device is placed on a hot plate set to
37 °C and a 25 g metal weight is placed over the sample
slider to ensure even heating and consistent mixing of
reagents (Fig.  2a). After 30  min, the device is removed
from the hot plate and the sample slider is pulled down to
position 2, placing it in contact with the dry dilution pads
and the weight is placed over top of it (Fig.  2b). After
10 min, the slider and weight are moved to position 3 in
contact with the wet dilution pads for ten more minutes
Cordray and Richards‑Kortum Malar J (2015) 14:472 Page 5 of 8

Fig. 2  Device in operation. For clarity the weight which would rest over the location of the sample pad has been omitted and the edges of the
sample and running buffer sliders have been outlined. a Sample slider at position 1, mixing RPA reagents and target and allowing amplification to
occur (30 min). b After amplification, the sample slider pulled down to position 2, in contact with dry dilution pads to absorb RPA buffer (10 min). c
The sample slider pulled down to position 3, in contact with wet dilution pads, for dilution with TBST (10 min). d Sample slider pulled down to posi‑
tion 4 into contact with lateral flow strip. The running buffer slider is pulled down to activate the strip (5 min)

(Fig. 2c). Finally, the sample pad is moved down to posi-
tion 4 over top of the lateral flow strip, the running buffer
slider is also pulled down to the strip, and the weight is
placed on top (Fig.  2d). The strip is allowed to run for
5 min, and then the device is scanned.
To test and optimize the dilution zone, 0.25 % Ponceau
S dye was used in place of sample to monitor the move-
ment of fluid through the system. All other components
were loaded as if an amplification reaction was to be run.
After amplification and dilution, rather than running the
lateral flow strip, the device was cut open and the sample
pad was removed and placed in 90 µL of TBST, vortexed,
and the concentration of dye in solution was measured
using a Cary 50 Spectrophotometer.
Results
The Plasmodium primers produced the expected primary
and secondary amplicons based on agarose gel analysis of
the products (Fig.  3a) and achieved a limit of detection
(LOD) of 5  copies/µL (50 total copies) using visual
inspection of the lateral flow strips (Fig.  3b). The visual
result was confirmed by using SBR to objectively deter-
mine if a strip was positive or not. A strip was consid-
ered positive if it had a SBR that was greater than three
standard deviations above the averaged SBR of negative
control strips. Using this cutoff, the limit of detection of
the assay, at which all samples were positive in every test,
was also 5 copies/µL (50 total copies).
Using dye to track the flow of fluids, the slider sys-
tem was capable of reliably moving fluids through the
system and transferring them to subsequent pads. The
number of dilution pads, their size, the amount of buffer
they were loaded with and the time spent on each pad
were all varied as experimental conditions. After opti-
mization of the dilution system, two pairs of blotter
paper pads were used, one wet and one dry, to achieve
the necessary dilution. The dry dilution pads were used
to remove some fluid on the sample pad so that it could
Cordray and Richards‑Kortum Malar J (2015) 14:472
a Copies/µL No
200 50 5 Target
650
600
550
500
450
400
350
300
250
200
150
100
Fig. 3  Performance of RPA nfo reaction and Plasmodium primers and probes in benchtop reactions. a An agarose gel stained with ethidium bro‑
mide showing the amplification of the primary (218 bp) and secondary (181 bp) products. b An example of the products run on lateral flow strips.
Note that the positive result at 5 copies/µL of target is easier to determine on the strips than on the gel
be replaced with TBST at the wet dilution pads. It was
found that by letting the fluids exchange for 10  min on
each set of pads under a 25  g weight, a final dilution of
1:49 (2.04 % ± 0.26 % dilution, n = 5) could be achieved.
No false positives due to lack of RPA buffer dilution were
observed using this design.
The total run time for the device is 55  min (30  min
incubation, 20  min dilution and 5  min lateral flow
detection). Including the loading and operation of the
device, the entire assay can be carried out in about an
hour. To test the performance of the system, the same
samples were simultaneously run side-by-side using
both conventional bench top methods and the paper
and plastic devices. Both methods were found to have a
limit of detection of 5  copies/µL (50 total copies) using
visual inspection and objective analysis by SBR (Fig.  4).
Although the limit of detection was unaffected, the
devices consistently had a lower SBR than the assay run
on the bench top. At both 200 and 50  copies/µL there
was approximately a 30 % reduction in the average SBR.
There was less than a 5 % difference at both 5 copies/µL
and in the no target controls. The reduced SBR at higher
target concentrations may be due to two factors: running
the assay in a paper matrix may reduce the mixing in the
system compared to running it in a tube. Additionally,
it was found that the plastic layered overtop of the lat-
eral flow strip increases the signal over both the test line
and the background of the strip driving the SBR closer
to unity.
Page 6 of 8
Detecon Test
b Line Line
Copies/µL
200
50
5
No Target
Discussion
A novel set of primers have been developed which
detects a sequence shared amongst the major human
infectious species of Plasmodium and which can detect
at least 5 copies/µL (50 total copies) of a plasmid contain-
ing a synthetic copy of a Plasmodium gene. This detect-
able concentration is comparable to other isothermal
amplification methods for malaria and below the LOD
of thin smear microscopy, the gold standard for malaria
diagnosis [16, 32]. These primers are used with RPA nfo,
an isothermal amplification method which can run at
relatively low temperatures, which produces results in as
few as 35  min when run on the bench top. A commer-
cially available assay for malaria which uses the LAMP
isothermal amplification technique has been developed
which is capable of detecting around 2 copies/µL (5 total
copies) [26, 27]. Although the RPA nfo reaction detects
fewer copies, the concentration at which it becomes posi-
tive is on the same order as LAMP and other isothermal
techniques, and RPA nfo may offer advantages such as a
shorter amplification run time and a readout on lateral
flow strips [16].
A paper and plastic device was created which is capable
of running the amplification reaction, diluting the ampli-
fied product by a repeatable amount and running the
results on an included lateral flow dipstick. The device
is made of simple components, can be assembled by the
user and uses a novel slider method to transport reagents
through the system. The equipment needed beyond the
Cordray and Richards‑Kortum Malar J (2015) 14:472
a Test Control b 3.2
Line Line Copies/µL
200
SBR
50
5
No Target
Fig. 4  Demonstration of performance of paper and plastic device. a Representative images of the lateral flow strips run in the devices. b Compari‑
son of the averaged SBR of samples run in the device and in tubes (n = 3), the error bars represent ±1 standard deviation. Strips were positive by
SBR if they were more than 3 standard deviations above the averaged no target control SBR, shown as a dotted line on the figure
device itself and the sample to be tested are a hot plate
capable of 37  °C, a reusable 25  g metal weight, the RPA
master mix, the running/dilution buffer (TBST) and
pipettes to load these reagents onto the device dilution,
running buffer, RPA and sample pads. This device runs
the entire assay, including detection, in around an hour
and has a limit of detection equivalent to when the assay
is run using conventional methods on the bench top.
Excluding the cost of the lateral flow strip and the RPA
nfo reaction pellet, the device costs around $1.05. The
commercial lateral flow strips cost around $3.14 each,
and the primers and RPA nfo reaction kits together cost
approximately $4.09. This gives an approximate total
reaction cost of $8.28 per test, most of which is due to
the cost of the commercial components rather than the
paper and plastic device.
A key limitation of the current work is that while a
self-contained system for running the reaction was pre-
sented, it requires the user to add set amounts of fluid
to various pads before sealing and operating the device.
This means that the system requires roughly the same
amount of manipulation as running the reaction on the
bench top. Future work could reduce the external parts
needed by integrating buffer and primer storage into the
device, perhaps by drying them on the pads. In addition,
the dilution system used increases the runtime of the
assay by 20 min. Although much of this time is hands-off,
future work should increase the run speed of the device
by developing a more efficient method for diluting the
sample. A study should be also be conducted to ensure
that the seal of the device is adequate, and that aerosols
cannot escape from the device in sufficient quantity to
become a source of future contamination.
This study was aimed at developing a proof-of-con-
cept device and used synthetic targets. In order for the
Page 7 of 8
2.8
2.4
2
1.6
1.2
0.8
1 10 100 1000
Starng Target Concentraon (#/uL) [log scale]
Device Tube
paper and plastic devices to be more useful in real world
settings, the assay should be tested and optimized on
infected and un-infected blood samples. Clinical samples
will offer a number of additional challenges compared
to the synthetic targets used in this study such as back-
ground human DNA and other blood components. Sam-
ple preparation techniques capable of lysing blood and
extracting DNA in a manner compatible with the RPA
nfo assay will need to be developed. Recently, there have
been a variety of studies which have examined meth-
ods to run PCR and isothermal amplification on clinical
malaria samples with minimal need for extra processing
or equipment by using for example, a simple lysis tech-
nique such as detergents or heat, adapting the amplifica-
tion protocol to reduce the inhibitory effects of blood,
and/or packaging the lysis and amplification together in
a matrix such as a hydrogel [33–35]. Once a suitable sam-
ple preparation method has been determined, further
work should be done to test the paper and plastic device
on clinical samples, and, if possible, to continue to add
more functions, such as the sample preparation directly
to the device.
Conclusion
A novel set of primers was developed which amplify a
sequence which is common to the human infectious spe-
cies of Plasmodium and operate an isothermal ampli-
fication reaction which is rapid and has an easy visual
readout. A paper and plastic device was also developed
which carries out the amplification of the samples, dilutes
the product and runs the result on a lateral flow strip.
When tested on synthetic targets, a limit of detection of
5  copies/µL (50 total copies) was found, which matches
the performance of the same assay run on the bench top.
This device is self-sealing and self-contained once wet
Cordray and Richards‑Kortum Malar J (2015) 14:472
reagents are added and needs only a 37  °C heat source,
reusable weight and timer. This device is a step towards
a fully self-contained and easy to operate low-resource
appropriate nucleic acid test for malaria diagnosis.
Authors’ contributions
MSC carried out the experimental work and drafted the manuscript. RRK
conceived of the study, participated in its design and coordination and helped
review and edit the manuscript. All authors read and approved the final
manuscript.
Acknowledgements
Funded by a grant from the Bill and Melinda Gates Foundation through the
Grand Challenges in Global Health Initiative.
Competing interests
The authors declare that they have no competing interests.
Received: 28 March 2015 Accepted: 18 November 2015
References
1. Niemz A, Ferguson TM, Boyle DS. Point-of-care nucleic acid testing for
infectious diseases. Trends Biotechnol. 2011;29:240–50.
2. Weigl B, Domingo G, LaBarre P, Gerlach J. Towards non-and minimally
instrumented, microfluidics-based diagnostic devices. Lab Chip.
2008;8:1999–2014.
3. Khairnar K, Martin D, Lau R, Ralevski F, Pillai DR. Multiplex real-time quan‑
titative PCR, microscopy and rapid diagnostic immuno-chromatographic
tests for the detection of Plasmodium spp: performance, limit of detec‑
tion analysis and quality assurance. Malar J. 2009;8:284.
4. Johnston SP, Pieniazek NJ, Xayavong MV, Slemenda SB, Wilkins PP, da Silva
AJ. PCR as a confirmatory technique for laboratory diagnosis of malaria. J
Clin Microbiol. 2006;44:1087–9.
5. Hänscheid T, Grobusch MP. How useful is PCR in the diagnosis of malaria?
Trends Parasitol. 2002;18:395–8.
6. Kuhn H, Demidov VV, Frank-Kamenetskii MD. Rolling-circle amplification
under topological constraints. Nucleic Acids Res. 2002;30:574–80.
7. Schweitzer B, Kingsmore S. Combining nucleic acid amplification and
detection. Curr Opin Biotechnol. 2001;12:21–7.
8. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N,
et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res.
2000;28:E63.
9. Compton J. Nucleic acid sequence-based amplification. Nature.
1991;350:91–2.
10. Euler M, Wang Y, Otto P, Tomaso H, Escudero R, Anda P, et al. Recombinase
polymerase amplification assay for rapid detection of Francisella tularen-
sis. J Clin Microbiol. 2012;50:2234–8.
11. Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using
recombination proteins. PLoS Biol. 2006;4:e204.
12. Jaroenram W, Owens L. Recombinase polymerase amplification com‑
bined with a lateral flow dipstick for discriminating between infectious
Penaeus stylirostris densovirus and virus-related sequences in shrimp
genome. J Virol Methods. 2014;208:144–51.
13. Mao X, Huang TJ. Microfluidic diagnostics for the developing world. Lab
Chip. 2012;12:1412–6.
14. Fu E, Ramsey SA, Kauffman P, Lutz B, Yager P. Transport in two-dimen‑
sional paper networks. Microfluid Nanofluidics. 2011;10:29–35.
15. Rohrman BA, Richards-Kortum RR. A paper and plastic device for per‑
forming recombinase polymerase amplification of HIV DNA. Lab Chip.
2012;12:3082–8.
Page 8 of 8
16. Cordray MS, Richards-Kortum RR. Emerging nucleic acid-based tests for
point-of-care detection of malaria. Am J Trop Med Hyg. 2012;87:223–30.
17. Okell LC, Bousema T, Griffin JT, Ouédraogo AL, Ghani AC, Drakeley CJ.
Factors determining the occurrence of submicroscopic malaria infections
and their relevance for control. Nat Commun. 2012;3:1237.
18. Okell LC, Ghani AC, Lyons E, Drakeley CJ. Submicroscopic infection in
Plasmodium falciparum-endemic populations: a systematic review and
meta-analysis. J Infect Dis. 2009;200:1509–17.
19. Cotter C, Sturrock HJ, Hsiang MS, Liu J, Phillips AA, Hwang J, et al. The
changing epidemiology of malaria elimination: new strategies for new
challenges. Lancet. 2013;382:900–11.
20. Kersting S, Rausch V, Bier FF, von Nickisch-Rosenegk M. Rapid detection
of Plasmodium falciparum with isothermal recombinase polymerase
amplification and lateral flow analysis. Malar J. 2014;13:99.
21. LaBarre P, Gerlach J, Wilmoth J, Beddoe A, Singleton J, Weigl B. Non-instru‑
mented nucleic acid amplification (NINA): instrument-free molecular
malaria diagnostics for low-resource settings. In: Engineering in Medicine
and Biology Society (EMBC), 2010 annual international conference of the
IEEE; 2010. p. 1097–9.
22. Marangi M, Di Tullio R, Mens P, Martinelli D, Fazio V, Angarano G, et al.
Prevalence of Plasmodium spp. in malaria asymptomatic African
migrants assessed by nucleic acid sequence based amplification. Malar J.
2009;8:12.
23. Paris DH, Imwong M, Faiz AM, Hasan M, Yunus EB, Silamut K, et al. Loop-
mediated isothermal PCR (LAMP) for the diagnosis of falciparum malaria.
Am J Trop Med Hyg. 2007;77:972–6.
24. Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko
H, et al. Detection of four Plasmodium species by genus- and species-
specific loop-mediated isothermal amplification for clinical diagnosis. J
Clin Microbiol. 2007;45:2521–8.
25. Hsiang MS, Greenhouse B, Rosenthal PJ. Point of care testing for malaria
using LAMP, loop mediated isothermal amplification. J Infect Dis.
2014;210:1167–9.
26. Polley SD, González IJ, Mohamed D, Daly R, Bowers K, Watson J, Mewse
E, Armstrong M, Gray C, Perkins MD. Clinical evaluation of a loop-
mediated amplification kit for diagnosis of imported malaria. J Infect Dis.
2013;208:637–44.
27. Hopkins H, González IJ, Polley SD, Angutoko P, Ategeka J, Asiimwe C,
Agaba B, Kyabayinze DJ, Sutherland CJ, Perkins MD. Highly sensitive
detection of malaria parasitemia in a malaria-endemic setting: perfor‑
mance of a new loop-mediated isothermal amplification kit in a remote
clinic in Uganda. J Infect Dis. 2013;208:645–52.
28. Tahar R, Basco LK. Detection of Plasmodium ovale malaria parasites
by species-specific 18S rRNA gene amplification. Mol Cell Probes.
1997;11:389–95.
29. Das A, Holloway B, Collins WE, Shama VP, Ghosh SK, Sinha S, et al. Species-
specific 18S rRNA gene amplification for the detection of P. falciparum
and P. vivax malaria parasites. Mol Cell Probes. 1995;9:161–5.
30. Green MR, Sambrook J. Molecular cloning: a laboratory manual. New York:
Cold Spring Harbor Laboratory Press; 2012.
31. Rohrman BA, Leautaud V, Molyneux E, Richards-Kortum RR. A lateral
flow assay for quantitative detection of amplified HIV-1 RNA. PLoS One.
2012;7:e45611.
32. Wongsrichanalai C, Barcus MJ, Muth S, Sutamihardja A, Wernsdorfer WH.
A review of malaria diagnostic tools: microscopy and rapid diagnostic
test (RDT). Am J Trop Med Hyg. 2007;77:119–27.
33. Taylor BJ, Howell A, Martin KA, Manage DP, Gordy W, Campbell SD, et al. A
lab-on-chip for malaria diagnosis and surveillance. Malar J. 2014;13:179.
34. Taylor BJ, Martin KA, Arango E, Agudelo OM, Maestre A, Yanow SK. Real-
time PCR detection of Plasmodium directly from whole blood and filter
paper samples. Malar J. 2011;10:244.
35. Port JR, Nguetse C, Adukpo S, Velavan TP. A reliable and rapid method for
molecular detection of malarial parasites using microwave irradiation and
loop mediated isothermal amplification. Malar J. 2014;13:454.